Inhibitor-sensitive FGFR2 and FGFR3 mutations in lung squamous cell carcinoma

Abstract

A comprehensive description of genomic alterations in lung squamous cell carcinoma (lung SCC) has recently been reported, enabling the identification of genomic events that contribute to the oncogenesis of this disease. In lung SCC, one of the most frequently altered receptor tyrosine kinase families is the fibroblast growth factor receptor (FGFR) family, with amplification or mutation observed in all four family members. Here, we describe the oncogenic nature of mutations observed in FGFR2 and FGFR3, each of which are observed in 3% of samples, for a mutation rate of 6% across both genes. Using cell culture and xenograft models, we show that several of these mutations drive cellular transformation. Transformation can be reversed by small-molecule FGFR inhibitors currently being developed for clinical use. We also show that mutations in the extracellular domains of FGFR2 lead to constitutive FGFR dimerization. In addition, we report a patient with an FGFR2-mutated oral SCC who responded to the multitargeted tyrosine kinase inhibitor pazopanib. These findings provide new insights into driving oncogenic events in a subset of lung squamous cancers, and recommend future clinical studies with FGFR inhibitors in patients with lung and head and neck SCC.

Figure 1

Recurrent mutations in FGFR2 and FGFR3 are observed in lung squamous cell carcinoma. (A) Sequencing data from TCGA were analyzed and recurrent mutations were observed in FGFR2 and FGFR3. The mutation S320C in FGFR2, in red, is located in the alternatively spliced exon in the IG-3 domain of FGFR2 IIIb; the remaining mutations are annotated to the IIIc isoform. FGFR3 mutations are annotated in the IIIc isoform. (B) Co-occurring somatic copy number alterations and mutations in samples with mutation.

Figure 2

A subset of lung SqCC mutations in FGFR2 and FGFR3 are transforming in anchorage independent growth assays and xenograft assays. (A) Colony formation compared to wild type in NIH-3T3 cells expressing FGFR mutations was calculated for each isoform and graphed. EGFR insNPG was included as a positive control, and the pBabe-puro Gateway empty vector (pBp GW) was included as a negative control. P-values were calculated with the student’s t-test and significance is indicated by asterisks; * < 0.05, ** < 0.01, *** < 0.001. (B) Nude mice injected with transforming FGFR2 mutant cells from (A) developed tumors, which were treated with BGJ398 (dashed lines) or vehicle (solid lines). (C) Tumors were dissected from the mice for visual inspection comparing treatment with vehicle or drug. Top panel, FGFR2-W290C tumors; bottom panel, FGFR2-S320C. Tumor images corresponding to FGFR2-K660N and FGFR2-WT tumors are shown in Figure S2.

Figure 3

Anchorage independent colony formation is abrogated in the presence of anti-FGFR inhibitors. (A) NIH-3T3 cells expressing each transforming mutation were seeded in the presence of increasing concentrations of ponatinib (AP24534) (left panel) and BJG398 (right panel). (B) Cells were serum starved and exposed to the indicated concentrations of ponatinib for four hours and then ligand stimulated for 30 minutes with FGF1, after which cells were lysed and probed via immunoblot. These experiments were performed with several other clinical inhibitors; those results are documented in Figure S4.

Figure 4

Ba/F3 cells dependent on FGFR signaling are sensitive to FGFR inhibitors. (A) Ba/F3 cells dependent on FGFR signaling were isolated by exchanging IL-3 with FGF-7 or FGF-9 and heparin. These cells were lysed and probed for FGFR2 or FGFR3 expression, phospho-FGFR, FRS2, and phospho-FRS2 Y436. Actin was used as a loading control. (B) Ba/F3 cells expressing each mutation construct were seeded into 96-well plates, in the presence of increasing concentrations of ponatinib (left panel) or BGJ398 (right panel). After four days, proliferation was measured with Cell Titer Glo. (C) IC₅₀ values were calculated for each mutation. These experiments were performed with other FGFR inhibitors; those results are documented Figure S5.

Figure 5

An oral squamous cell carcinoma patient harboring a somatic FGFR2 P253R mutation demonstrates a partial response to an FGFR inhibitor. (A) A schematic shows the P253R mutation in the FGFR2 extracellular domain. (B) mRNA sequencing was performed and a somatic mutation in FGFR2 was identified, shown in the IGV viewer. (C) Pre- and post-treatment images from the patient.